Down, But Not Out: Partial Elimination of Androgen Receptors in the Male Mouse Brain Does Not Affect Androgenic Regulation of Anxiety or HPA Activity

Abstract

We previously found that androgen receptor (AR) activity mediates two effects of T in adult male mice: reduction of anxiety-like behaviors and dampening of the hypothalamic-pituitary-adrenal response to stress. To determine whether brain ARs mediate these effects, we used the Cre/loxP technology seeking to disable AR throughout the central nervous system (CNS). Female mice carrying the floxed AR allele (ARlox) were crossed with males carrying cre recombinase transgene controlled by the nestin promoter (NesCre), producing cre in developing neurons and glia. Among male offspring, four genotypes resulted: males carrying ARlox and NesCre (NesARko), and three control groups (wild types, NesCre, and ARlox). Reporter mice indicated ubiquitous Cre expression throughout the CNS. Nevertheless, AR immunocytochemistry in NesARko mice revealed efficient knockout (KO) of AR in some brain regions (hippocampus and medial prefrontal cortex [mPFC]), but not others. Substantial AR protein was seen in the amygdala and hypothalamus among other regions, whereas negligible AR remained in others like the bed nucleus of the stria terminalis and dorsal periaqueductal gray. This selective KO allowed for testing the role of AR in hippocampus and mPFC. Males were castrated and implanted with T at postnatal day 60 before testing on postnatal day 90-100. In contrast with males with global KO of AR, T still modulated anxiety-related behavior and hypothalamic-pituitary-adrenal activity in NesARko males. These results leave open the possibility that AR acting in the CNS mediates these effects of T, but demonstrate that AR is not required in the hippocampus or mPFC for T's anxiolytic effects.

Figure 1.. AR immunoreactivity is abolished in the hippocampus and mPFC of NesARko mice.

Mice with NestinCre and the LacZ reporter transgene displayed LacZ activity throughout the brain, including the hippocampus (A, inset of CA1 at higher magnification) and mPFC (B, inset from prelimbic cortex), indicating widespread Cre expression. Immunohistochemical staining for AR in positive control WT males showed normal AR-ir in all expected brain areas including the hippocampus (C) and mPFC (D). In NesARko males, immunohistochemical staining indicated complete absence of AR protein in both the hippocampus (E) and mPFC (F). As a negative control, iTfm mice with global recombination of the AR allele showed no AR-ir in the same areas (G and H). Black scale bar = 200 μm; white scale bar = 40 μm. Abbreviations: dg, dentate gyrus; fmi, forceps minor of the corpus callosum; Cg1, cingulate cortex area 1.

Figure 2.. NesARko only partially knocked down AR in some brain regions.

Mice with NestinCre and a LacZ reporter transgene showed evidence of robust Cre expression throughout the brain, including the amygdala (A, inset of ceAMY) and PVN (B, inset of parvocellular division). In WT males, these brain regions (C and D) display abundant AR-positive cells as revealed by immunohistochemistry. However, in NesARko males, immunohistochemical staining demonstrated residual expression of AR protein in these two brain regions (E and F), indicating AR was only knocked down, not knocked out, in these brain regions. As expected, negative control iTfm mice with global recombination of the AR allele showed no AR-ir in these regions (G and H) or anywhere else. Black scale bar = 200 μm; white scale bar = 40 μm. Abbrevaitions: opt, optic tract; bla, basolateral amygdala; 3v, third ventricle.

Figure 3.. Anxiety-related behavior in WT, NesARko, ARlox, and NesCre male mice: (A) number of rears; (B) time spent in the center area; (C) number of head dips; (D) time spent in the open arm; (E) time spent with the novel object; (F) number of visits to the novel object; (G) number of light side entries and time spent in the light side when the L/D box test was run after and before the EPM test.

For most tests, NesARko males did not differ from WT males in anxiety-related behavior. However, WT males performed more head dips than did NesARko or NesCre mice in the EPM (C) and more rears (A) in the OF than NesARko males, suggesting an unexpected effect of the Cre transgene alone on those behaviors. *, P < .05.

Figure 4.. HPA response to L/D box exposure in WT, NesARko, ARlox, and NesCre male mice.

No group differences were found in basal corticosterone (CORT) levels, nor in the response and recovery of HPA activity after exposure to the anxiety-provoking L/D box, indicating that AR in the hippocampus and mPFC are not necessary for T's modulatory effects on HPA response. All CORT levels significantly differed from each other at all time points (P < .05), except times 0 and 120. *, similar CORT levels at baseline and 120 min across groups.